P-Link: A method for generating multicomponent cytochrome P450 fusions with variable linker length

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Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P-LinK),. which was validated by fusing P450(cin) monooxygenase (CinA) to the flavodoxin shuttle protein (CinC). CinC was fused to the C terminus of CinA through a series of 16 amino acid linkers of different lengths in a single experiment employing 3 PCR amplifications. Screening for 2-beta-hydroxy-1,8-cineole production by CinA-CinC fusion proteins revealed that enzymatically active variants possessed linker lengths of more than 5 amino acids, reaching optimum enzyme activity at a linker length of 10 amino acids. Our P-LinK method not only minimizes experimental effort and significantly reduces time demands but also requires only a single cloning and transformation step in order to generate multiple linker variants (1 to 16 amino acids long), making the approach technically simple and robust.
Original languageEnglish
JournalBioTechniques
Volume57
Issue number1
Pages (from-to)13-20
ISSN0736-6205
DOIs
Publication statusPublished - 2014
CitationsWeb of Science® Times Cited: No match on DOI

    Research areas

  • cinAgene, cinCgene, 2-beta-hydroxy-1,8-cineole, amino acid linkers, CinA protein C terminus, monooxygenase, CinA-CinC fusion protein, CinC flavodoxin shuttle protein, cytochrome P450 9035-51-2 EC 1.14.14.1, fusion proteins, 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines, 10064, Biochemistry studies - Proteins, peptides and amino acids, 10802, Enzymes - General and comparative studies: coenzymes, Biochemistry and Molecular Biophysics, cloning laboratory techniques, genetic techniques, P-Link laboratory techniques, genetic techniques, PCR amplification polymerase chain reaction amplification laboratory techniques, genetic techniques, transformation laboratory techniques, genetic techniques, Enzymology, Methods and Techniques, BIOCHEMICAL, BIOCHEMISTRY, ESCHERICHIA-COLI, ELECTRON-TRANSFER, REDUCTASE DOMAIN, REDOX PARTNER, P450 ENZYMES, HEME DOMAINS, PROTEIN, EXPRESSION, SYSTEM, 1,8-CINEOLE, protein engineering, directed evolution, monooxygenase, multicomponent system, fusion protein, P450(cin), linker, PLICing

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